Endothelial Dysfunction in Youth-Onset Type 2 Diabetes: A Clinical Translational Study

BACKGROUND: Youth-onset type 2 diabetes (Y-T2D) is associated with increased risk for coronary atherosclerotic disease, but the timing of the earliest pathological features and evidence of cardiac endothelial dysfunction have not been evaluated in this population. Endothelial function magnetic resonance imaging may detect early and direct endothelial dysfunction in the absence of classical risk factors (severe hyperglycemia, hypertension, and hyperlipidemia). Using endothelial function magnetic resonance imaging, we evaluated peripheral and coronary artery structure and endothelial function in young adults with Y-T2D diagnosed ≤5 years compared with age-matched healthy peers. We isolated and characterized plasma-derived small extracellular vesicles and evaluated their effects on inflammatory and signaling biomarkers in healthy human coronary artery endothelial cells to validate the imaging findings. METHODS: Right coronary wall thickness, coronary artery flow–mediated dilation, and brachial artery flow–mediated dilation were measured at baseline and during isometric handgrip exercise using a 3.0T magnetic resonance imaging. Human coronary artery endothelial cells were treated with Y-T2D plasma–derived small extracellular vesicles. Protein expression was measured by Western blot analysis, oxidative stress was measured using the redox-sensitive probe dihydroethidium, and nitric oxide levels were measured by 4-amino-5-methylamino-2’,7’-difluororescein diacetate. RESULTS: Y-T2D (n=20) had higher hemoglobin A1c and high-sensitivity C-reactive protein, but similar total and LDL (low-density lipoprotein)-cholesterol compared with healthy peers (n=16). Y-T2D had greater coronary wall thickness (1.33±0.13 versus 1.22±0.13 mm; P=0.04) and impaired endothelial function: lower coronary artery flow–mediated dilation (−3.1±15.5 versus 15.9±17.3%; P<0.01) and brachial artery flow–mediated dilation (6.7±14.7 versus 26.4±15.2%; P=0.001). Y-T2D plasma–derived small extracellular vesicles reduced phosphorylated endothelial nitric oxide synthase expression and nitric oxide levels, increased reactive oxygen species production, and elevated ICAM (intercellular adhesion molecule)–mediated inflammatory pathways in human coronary artery endothelial cells. CONCLUSIONS: Coronary and brachial endothelial dysfunction was evident in Y-T2D who were within 5 years of diagnosis and did not have severe hyperglycemia or dyslipidemia. Plasma-derived small extracellular vesicles induced markers of endothelial dysfunction, which corroborated accelerated subclinical coronary atherosclerosis as an early feature in Y-T2D. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02830308 and NCT01399385.


Characterization of Small Extracellular Vesicles by Transmission Electron Microscopy (TEM):
A 400 mesh Formvar®/carbon-coated copper grid was soaked in 100% ethanol for 1 minute.A 10 µL droplet of small extracellular vesicle samples (in phosphate buffered saline [PBS]) was placed on a sheet of Parafilm, and the grid was floated with the dark-coated side facing the sample, incubating for 90 minutes.The residual sample was washed from the grid by dipping it into 20 µL drops of molecular biology-grade water three times.The grids were then stained for 1 minute with 2% uranyl acetate and allowed to dry for 60 minutes before imaging with a JEM-1200 microscope.

Immunogold Labeling and Transmission Electron Microscopy (TEM) of Small Extracellular Vesicles:
Approximately 10 x 10^9 YD-Ex (as quantified by NTA) were incubated with anti-CD9 and anti-CD63 antibodies for 1 hour at room temperature.A nonspecific IgG isotype antibody served as a negative control.Samples were diluted 1:10 in PBS and incubated with 10.4-nm protein A gold-conjugated secondary anti-rabbit antibody (Nanoprobes, NY, USA) for one hour, followed by crosslinking with 1% glutaraldehyde for 15 minutes, both at room temperature.Sample volumes were brought to 500 µL then filtered through Amicon Ultra 0.5 mL centrifugal filters, MWCO 100 kDa (Millipore Sigma, MO, USA) for buffer exchange according to the manufacturer's recommendations.TEM grids (ultra-thin carbon film supported by Lacey carbon grid 300 mesh, Ted Pella, CA, USA) were glow-discharged (PELCO easiglow, Ted Pella), CIRCRES/2024/324272D/R2 Endothelial Dysfunction in Youth-Onset Type 2 Diabetes: A Clinical Translational Study Abd-Elmoniem KZ et al.
applying a current of 35 mA for 60 seconds.After immunogold labeling and chemical crosslinking, 3 µL of purified small extracellular vesicle samples were applied to the glow-discharged TEM grid.The sample was incubated on the grid for 60 seconds for absorption onto the carbon, and excess liquid was blotted using Whatman filter paper No. 1, followed by staining with a 1% phosphotungstic acid solution for 60 seconds.The negatively stained sample was mounted on a room-temperature side-entry holder and loaded into a Tecnai F20 TEM (FEI, OR, USA) equipped with a K2 detector (Gatan, CA, USA).The microscope was operated in low gain mode, and images were acquired with a nominal magnification of 25,000 (corresponding to a pixel size of 1.52 Å / pixel) for a total of 8 seconds (200 ms per frame) with a total dose of 150 electrons / Å^2.Beam-induced motion and stage drift were corrected by whole frame alignments using the MotionCor software tool, bandpass filtered (10 and 200 Å) for visualization purposes and finally analyzed in ImageJ 1.49v (Bethesda, NIH, USA).

Immunoblotting Analysis of Small extracellular vesicle-Specific Markers:
To investigate the presence of small extracellular vesicle protein markers, immunoblotting analysis was performed using intact small extracellular vesicle samples.Determination of small extracellular vesicle concentrations was by a Micro-BCA assay kit (Pierce, Rockford, IL, USA) in accordance with the manufacturer's instructions.The total protein content was calculated by multiplying the protein concentration by the volume of the isolated small extracellular vesicles.
Intact small extracellular vesicle samples isolated using the kit method were directly mixed with an equal volume of 2× Laemmli sample buffer.From each prepared sample, 16 µg of protein lysates were separated by 4-12% SDS-PAGE and blotted onto Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA).Membranes were blocked with 5% skim CIRCRES/2024/324272D/R2 Endothelial Dysfunction in Youth-Onset Type 2 Diabetes: A Clinical Translational Study Abd-Elmoniem KZ et al. milk in PBS containing 0.05% Tween-20 and probed with primary antibodies overnight at 4°C.The antibodies used for immunoblotting were small extracellular vesicle-specific anti-TSG101, anti-CD9, and anti-CD63; Anti-ApoE was used to confirm the absence of lipoproteins potentially included in the extraction process; and anti-calnexin, a non-small extracellular vesicle endoplasmic reticulum-related antibody, was used as per Minimum information for studies of extracellular vesicles 2018 guidelines 25 .After the membranes were washed three times with PBS containing 0.05% Tween-20 (PBST) for 10 minutes, they were incubated for 1 hour with goat anti-rabbit IgG and HRP-conjugated antibody (#7074; 1:3000; Cell Signaling Technology, Danvers, MA, USA).All membranes were washed three times for 10 minutes with PBST.The signals of the HRP-conjugated antibodies were developed using Western blotting detection reagent (Western Femto ECL Kit, Invitrogen, USA) and visualized using a chemiluminescence detection system (ChemiDoc Bio-Rad, Hercules, CA, USA).To further verify the purity of small extracellular vesicle isolation, immunoblotting analysis was performed on small extracellular vesicles from healthy donors and Y-T2D cell lysate from HCAEC was used as a negative control for purity analysis.

Human Coronary Artery Endothelial Cell Cultures
Primary HCAECs were cultured for a maximum of four passages in endothelial basal medium-2 (EBM-2) supplemented with growth factors [endothelial growth medium EGM and TM-2 MV Microvascular Endothelial Cell Growth Medium-2 BulletKit from Lonza (Walkersville, MD, USA) containing 2% serum].Cells were maintained at 37°C in a humidified incubator with 5% CO2 and 95% air.Primary HCAECs were seeded at a density of 70,000 cells/well in 12-well plates or 150,000 cells/well in 6-well plates for protein analysis, respectively.HCAEC donors (n CIRCRES/2024/324272D/R2 Endothelial Dysfunction in Youth-Onset Type 2 Diabetes: A Clinical Translational Study Abd-Elmoniem KZ et al.

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= 4) ranged in age from 19 to 32 years old and cells were purchased from Lonza and Lifeline (Frederick, MD, USA).Mycoplasma contamination testing was performed by Lonza and Lifeline.HCAECs were then cultured with the endothelial basal medium prepared in small extracellular vesicles depleted fetal bovine serum (FBS) purchased from (Gibco, Waltham, MA, USA).

Tracing Small extracellular vesicle Uptake in the Ex Vivo System of HCAECs
Small extracellular vesicle staining was conducted using SYTO-RNASelect dye (SYTO® RNASelect™ stain for RNA staining or BODIPY® TR ceramide for membrane staining, SYTO® RNASelect™ Green Fluorescent Cell Stain (S32703).HCAECs cultured in chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System), then 100 μL labeled small extracellular vesicles were diluted in 400 μL EBM2 endothelium growth medium and incubated with HCAECs for 24h and 48h.The HCAECs were then labeled with Phalloidin (CST, 8953) (1:200) at room temperature for 15 minutes, followed by staining with Prolong® Gold Antifade Reagent with DAPI.The cells were then visualized using a fluorescent microscope.

HCAEC Incubation and Western Blot Analysis of Nitric Oxide and Inflammatory Markers
Whole cell protein lysates were isolated from HCAECs treated with plasma-derived small extracellular vesicles from 11 plasma samples at a final concentration of 50 µg/mL or from PBScontrol untreated cells for 48 hours using RIPA buffer (Thermo Scientific Pierce, Rockford, IL, USA) supplemented with protease and phosphatase inhibitor cocktail (Thermo Scientific Pierce).

HCAEC Incubation and Measurement of Reactive Oxygen Species
HCAECs were seeded into 6-well plates and exposed to small extracellular vesicles from participants for 4 hours.Dihydroethidium (2.5 µM), a redox-sensitive fluorescent dye, was applied to cells for 30 minutes at 37°C.Cells were washed three times with warm PBS, EBM2 growth media was added, and cells were collected by scraping before analysis by flow cytometer (Fortessa, NHLBI, NIH, Bethesda, USA).Light scatter parameters were set to eliminate dead cells and subcellular debris.The red ethidium signal was measured, and oxidative stress was estimated using the mean fluorescence intensity (MFI) of the population.Auto-fluorescence gains were determined in unlabeled cells and set at the first logarithmic decade.

HCAEC Incubation and Measurement of Nitric Oxide
HCAECs were seeded into 6-well plates and exposed to small extracellular vesicles from participants for 48 hours.A NO-specific probe (DAF-FM DA) was applied to cells for 20-60 minutes at temperatures ranging from 4℃ to 37℃.HCAECs were not trypsinized.Cells were washed 3 times with warm PBS1X to remove excess probe.Fresh medium was then added to the HCAECs, and they were incubated for an additional 30 minutes to allow complete desiccation of the intracellular diacetates.MFI was measured at fluorescence excitation and emission of 488 and 530/30 nm respectively by flow cytometry.

Probing of Plasma-Derived Small extracellular vesicles Transfer to Target HCAECs
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PKH26 lipid dye was diluted in 100 µL diluent C to a final concentration of 8 µM (dye solution).
Then 10 µg of small extracellular vesiclesin 20 µL DPBS were diluted with 80 µL diluent C, added to the dye solution, and incubated for 5 minutes while mixed with gentle pipetting.Excess dye was bound with 100 µL 10% small extracellular vesicle-depleted fetal bovine serum (Sigma-Aldrich) in Dulbecco's modified Eagle's medium (Sigma-Aldrich) as previously described 28 .
Small extracellular vesicles were washed twice by centrifugation (13,000 g, 60 min).Interaction of PKH26-labeled small extracellular vesicles and target ECs was assessed by flow cytometry (Fortessa, Becton Dickinson, USA).

Nanoparticle Tracking Analysis
Nanoparticle tracking analysis (NTA) from Malvern (NanoSight NS300, Malvern Instruments, Malvern, USA) was used for size distribution and concentration measurements of small extracellular vesicle samples in liquid suspension from the properties of both light scattering and Brownian motion.The NanoSight NS300 with a 405-nm laser instrument (Malvern Instruments, Malvern, USA) was used to detect nanovesicles.Five videos of typically 60-second duration were taken.Data were analyzed using the FlowJo software.The Brownian motion of each particle was tracked using the Stokes-Einstein equation: D = kT/6πηr, where D is the diffusion coefficient, kT/6πηr = f0 is the frictional coefficient of the particle, for the special case of a spherical particle of radius r moving with uniform velocity in a continuous fluid of viscosity η, k is Boltzmann's constant, and T is the absolute temperature.

Characterization of plasma-derived small extracellular vesicles from healthy volunteers and Y-T2D.
Plasma-derived small extracellular vesicles were isolated from healthy lean and T2D volunteers.Western blot analysis was conducted on lysates sourced from the small extracellular vesicle fraction and cell pellet, utilizing antibodies against CD81 and Alix.CIRCRES/2024/324272D/R2 Endothelial Dysfunction in Youth-Onset Type 2 Diabetes: A Clinical Translational Study Abd-Elmoniem KZ et al.